PT1773890E - Process for the preparation of low molecular weight heparin - Google Patents

Abstract

The preparation process includes a depolymerisation of heparin by nitrosation which provides low molecular weight heparin with a nitrite and N-nitrosamine content within the limits imposed by the Farmacopea Europea IVth edition. The invention also relates to a process for preparing purified low molecular weight heparin. In particular the purification process is applied to low molecular weight heparin by a nitrosation process, in which elimination of the nitrose groups is performed by heating a water solution containing low molecular weight heparin at a pH comprised between 3.0 and 13.0 to a temperature comprised between 40°C and 90°C for a time comprised between 15 minutes and 180 minutes, or by treatment of a water solution containing low molecular weight heparin by radiation with microwaves at a frequency comprised between 900 and 2450 MHz at a power of 600-1000 Watts, for a time period of between 30 and 300 seconds.

Description

1

DESCRIPTION

" PROCEDURE FOR THE PREPARATION OF LOW MOLECULAR WEIGHT HEPARIN " Technical area Heparin and its derivatives are among the products most used in the prevention and treatment of thromboembolic events.

This active ingredient, heparin, has a very important effect on coagulation and is therefore used to prevent thrombotic events after surgery and in the treatment of pulmonary embolisms or deep venous thrombosis. The anticoagulant effect of heparin is so important that treatment with this drug requires constant monitoring of hemocoagulant processes, leading to patient hospitalization, or at least outpatient treatment at day hospital. Prior art The reduction of the molecular weight of heparin by fractionation (LO Andersson et al., Throm. Res. 9: 575 (1976)) or by depolymerization resulted in active ingredients that are less anticoagulant, although they continue to exhibit good antithrombotic activity residual.

For example, European patents EP 0014184, EP 0040144, EP 0076279 and EP 0121067 describe various depolymerization procedures for heparin and obtain various types of low molecular weight heparin, which is usually indicated by the acronym LMWH. These types of LMWH exhibit different molecular weights and chemical structures that are modified in a variable manner, especially in their terminal groups, as well as different pharmacological activities.

In particular, molecular weight reduction gives the depolymerized products a lower overall anticoagulant activity (APTT-activated partial thromboplastin time) when compared to the initial heparin, whereas Axa activity (anti-Xa activity) is maintained at good levels , since it is the antithrombotic activity of the product (FA Ofosu et al. " Mechanism of action of low molecular weight heparins and heparinoids " Ballicre's Clin Haematol 3: 505-529 (1990)). US 4,804,652 and 4,351,938 disclose procedures for the preparation of such LMWH and for its use as anticoagulant and antithrombotic. Pharmacodynamics is also positively affected by the reduction of molecular weight, since the active ingredient is maintained for longer in the blood circuit, with higher blood levels than unfractionated heparin (L. Bara et al., &Quot; Increased anti- Xa bioavailability for a low molecular weight heparin compared with unfractionated heparin! Semin. Thromb. Hem., 11: 316-317 (1985)).

The previously described characteristics for LMWH have led the skilled person in the search for procedures for their production using extremely varied depolymerization procedures of heparin. Procedures were used which exploit radical hydrolysis reactions (hydrogen peroxide and copper catalyst, or peracids) periodic oxidation hydrolysis reaction or enzymatic hydrolysis with heparinase, or β-elimination reactions by basic hydrolysis of the heparin ester, or treatments with sodium nitrite in an acidic environment. The last mentioned procedure, described and claimed in European patents EP 0014184 and EP 0076279, provides a LMWH which then requires further purification or fractionation. The use of sodium nitrite 3 provides depolymerized heparin with a molecular weight which is more or less low according to the amount of sodium nitrite used but which requires great care and attention because the final product should contain no more than a few parts per million (ppm) of nitrite ions and less than 0.25 ppm of N-nitroses (N-NO) groups.

If, on the one hand, the content of sodium nitrite in the final product (JC Lormeau et al., U.S. Patent 5,019,649 (1991) and its determination pose no problem, on the other hand the content and quantification of the groups N-NO are difficult to execute.

Since, in theory, the presence of secondary amines in the heparin structure should not be verified, the only functional groups capable of providing sufficiently stable N-nitrosated derivatives are the N-acetylglucosamine groups.

A valid hypothesis may lie in the fact that the N-nitrosated groups appearing as a few ppm in the depolymerized product prior to purification derive from a nitrosation of the N-acetylglucosamine present in the heparin.

The N-NO groups derived from the nitrosation of heparin are very stable, although this does not occur at the level of N-alkyl nitrosamines, and probably during the depolymerization reaction a part of the acetyl-N-NO-glucosamine formed can react, acetyl group and converting to the deacetylglucosamine diazonium salt, yielding the depolymerized heparin in the normal reaction.

As these N-NO groups are very stable, it is essential that a depolymerization procedure is applied which leads to an N-nitrosamine content which is below the prescribed limit or, alternatively, to eliminate any N-NO group present, with a view to meeting the prescriptive limits established by the European Pharmacopoeia Fourth Edition. 4

It is known that N-NO groups are particularly sensitive to high energy radiation, such as ultraviolet rays and γ rays, and that the treatment of the product (depolymerized heparin by nitrosation) in solution or even in the solid state with these radiation may lead to the total disappearance of nitrites and N-NO residues contained in the product.

UV rays are widely used for the maintenance of sterility in sterile water or for the reduction of nitrous oxide derivatives in drinking water. U.S. Patent 5,599,801 to J.F. Branellec et al. Discloses a procedure and apparatus for the removal of the nitrous groups of the low molecular weight heparins obtained with the use of the nitrosation procedure. The limitation of this procedure lies in the low penetration power of UVA rays, both in solutions and in solids: This has an effect on the times and apparatus used for purification. The stability of the product exposed to lightning treatment is the major problem with the treatment by γ rays.

For this reason, it is necessary to provide a procedure for reducing the amounts of nitrites and / or N-NO groups in LMWH, where the procedure is effective, more economical compared to the prior art, and very efficient.

One of the main objects of the present invention is to carry out a depolymerization procedure which allows the production of LMWH with residual N-NO levels of less than 0.25 ppm.

It is an object of the present invention to provide a procedure for depolymerization to obtain a LMWH with nitrite levels of less than 5 ppm.

Another object of the present invention is to provide a depolymerized heparin purification procedure, obtained by the use of sodium nitrite, capable of purifying the depolymerized heparin by reducing the amounts of residual nitrites and nitrosated N-NO groups.

These objects and others which will emerge from the detailed description set forth below, fall within the present invention, which has developed a procedure for the preparation of LMWH and a purification procedure thereof. The present invention also relates to a process for the preparation of LMWH having a level of nitrites and simultaneously a level of N-NO nitrous groups within the limits indicated by the European Pharmacopoeia, Fourth Edition, having the features set forth in claims 1-7 attached.

Another object of the present invention is to provide a purification procedure capable of obtaining a reduction of the nitrite and N-NO groups which may be present in the LMWH at levels below the limits given by the European Pharmacopoeia, Fourth Edition, by heat or microwave treatment. The European Pharmacopoeia, Fourth Edition, is incorporated herein by reference. In particular, page 1296, on heparins in general; pages 1297-1299 relating to LMWH; pages 1610-1612 for Nadroparin (depolymerized heparin obtained by nitrosation); pages 999-1000 for Dalteparin (depolymerized heparin obtained by nitrosation). 6

Description of the Invention The procedure of the present invention is carried out using a standard concentration of commercially available heparin composed of 1% to 25% (grams per heparin relative to the total weight of the solution); preferably from 5% to 20%; and more specifically preferably from 10% to 16%. Commercially available heparin is usually lyophilised as a sodium salt-heparin natricum. European Pharmacopoeia, Fourth Edition, page 1296. The Applicant has discovered that the depolymerization of heparin in the presence of sodium nitrite is effected with a weight-for-weight ratio , between heparin and sodium nitrite, 60: 1 and 45: 1.

Advantageously the Applicant has found that a weight: weight ratio of 60: 1 to 45: 1, between heparin and sodium nitrite, produces LMWH with a nitrite content of less than 5 ppm and a group content of N-NO less than 0.25 ppm.

In a preferred embodiment, the weight: weight ratio of heparin to sodium nitrite is 52: 1 to 47: 1.

In the above-mentioned weight ratios, sodium nitrite is considered as such regardless of the salt used; for example, sodium nitrite is pure by analysis, having a sodium nitrite degree of about 97%.

In use, the lyophilized form of sodium heparin is dissolved at, for example, 25 ° C in a container provided with means for heating and stirring.

Preferably, the depolymerization procedure is carried out at a temperature in the range of 15Â ° C to 30Â ° C, for example between 23Â ° C and 27Â ° C, preferably at 25Â ° C. To the solution is added sodium nitrite, for example 97% sodium nitrite, and an acidifying substance, for example hydrochloric acid, to bring the pH of the solution to a pH between 2.0 and 4.0. The disappearance of the nitrite is monitored with the use of starch-iodine paper. Subsequently, the pH of the solution is brought to between 10 and 13 by the addition of an alkaline substance. Finally, NaBH 4 is added in an amount by weight of 0.5% to 2.5%, based on the initial weight of the heparin. At this point, the solution is allowed to stand at between 20-30 ° C, for example 25 ° C, for a time comprised between 10 and 30 hours, for example 20 hours. Excess NaBH4 is removed by the addition of an acidic substance to a pH comprised between 2 and 4, for example pH 3.

Following the above procedure, a depolymerized product is obtained which complies with the physico-chemical and biological specifications of the European Pharmacopoeia, Fourth Edition, for depolymerized heparin with nitrous acid, which has an N-nitrosamine content of less than 0.25 ppm and a final amount never less than 70% (grams of depolymerized heparin relative to the starting weight of the heparin), for example an amount between 75% and 85%.

With a weight ratio of heparin sodium nitrite of less than 45: 1 a product is obtained with an N-nitrosamine content above the established limits and with a produced amount lower than desired. This product should be further purified. With a weight ratio between sodium heparin-nitrite of greater than 60: 1, no acceptable level of depolymerisation is obtained. The subject purification procedure of a depolymerized heparin obtained by nitrosation may be carried out by heat or microwave treatment.

It has been found that both heat and microwaves between 9900 and 2450 Megahertz (MHz) are able to drastically reduce the presence of nitrites and N-nitrosated groups with a pH of 3.0 to 13.0, preferably with a pH preferably from 4.0 to 12.0, for example with a pH of 10-12. Heat and microwaves may be used separately although microwave treatment of aqueous solutions containing LMWH leads to heating the aqueous solution and therefore to synergistic and microwave heating in the removal of the nitrous groups present in LMWH derived from depolymerization by nitrosation.

In one embodiment, the purification procedure is effected by heating.

An aqueous solution containing 1% to 20% (weight: weight) of LMWH (obtained by nitrosation) is heated and agitated for purification until a temperature of 40 ° C to 90 ° C is reached. preferably from 60 ° C to 80 ° C, with a pH of from 3.0 to 13.0, preferably from 4.0 to 12.0, for a period of from 0.5 to 3 hours.

The solution is then brought to a pH of from 11 to 13, preferably pH 12, at a temperature of from 70øC to 90øC, with stirring. The solution is purified by membrane ultrafiltration following known methods.

In one embodiment, the purification procedure is performed by the use of microwaves. Purification of LMWH containing nitrites and N-nitrosate groups may be effected by subjecting the aqueous solution containing 1% to 20% of LMWH at a pH of 7.0 to 13.0, preferably 10.0 to 20, 0 to microwave radiation for a time period comprised between 60 and 300 seconds, using microwaves with a frequency comprised between 900 and 2450 MHz, at power between 600 and 100 watts, in the presence of inert silicates belonging, preferably, to class of the montemorilonite, in the form of powder and in amounts of 5% to 15% relative to the weight of the LMWH. The presence of the inert silicate acts as an amplifier of the effect of the microwaves. The suspension containing LMWH in solution and inert silicate is cooled to room temperature, filtered to remove the inert material, the pH is brought to 6.5 to 7.0 the solution is ultrafiltered to eliminate the salts present in the solution and the active ingredient is lyophilized.

An extremely pure LMWH, as shown in Table 1, is obtained in amounts of 96% to 98%, and with substantially unchanged pharmacological activity.

Table 1: ANALYSIS OF SAMPLES BEFORE AND AFTER THE PHASE OF

PURIFICATION

(Ppm) N-NO (ppm) N-NO (ppm) Before After Before After Before After After Before Sample 1 (Example 2) 36, 93 31.18 124.46 128.14 2.57 0.12 0.13 0.05 Sample 2 (example 3) 36.93 38.57 124.46 118.35 2.57 nv * 0.13 0.07 Sample 3 (example 4) 38.93 32.61 144.46 152.74 0.79 n.v. * 0.18 0.03 Sample 4 (example 5) 39.23 33.18 107.33 100.11 0.38 n.v. * 0.21 0.08 Sample 5 (example 6) 39.23 36.64 107.33 11.64 0.38 n.v. * 0.16 n.v. * n.v. *: not evaluable

Analyzes of the biological parameters, i.e. the anti-IIa activity (Alia) and the anti-Xa activity (Axa), the chemical parameters, i.e. the content of the nitrites and the N-nitrosated groups, were performed with procedures 10 described in the European Pharmacopoeia, Fourth Edition, for LMWH, pages 999, 1297 and 1610.

As can be seen in Table 1, the depolymerization procedure of the present invention allows obtaining a LMWH with a nitrite amount of less than or equal to 5 ppm (as determined by liquid chromatography, European Pharmacopoeia, Fourth Edition, page 999) and an amount of nitrous groups less than or equal to 0.25 ppm (determined upon the disruption of the N-NO bond by the use of hydrobromic acid in ethyl acetate in a reflux condenser, measuring the NO release by chemiluminescence, European Pharmacopoeia , Fourth Edition, page 1611, known as the EPS solution).

In a preferred embodiment, the EPS solution can be subjected to the purification procedure of the present invention for the purpose of obtaining purified dalteparin or nadroparin.

Alternatively, in another embodiment, the EPS solution may be subjected to a separation in dalteparin or nadroparin. Subsequently, the fractions of dalteparin and nadroparin obtained from the separation are purified, with the aim of obtaining purified dalteparin or nadroparin.

The examples included herein should be considered as a further illustration of the invention and therefore are not intended to be limiting. EXAMPLE 1: Preparation of low molecular weight heparin by depolymerization using sodium nitrite

Dissolve 100 g of sodium heparin with anticoagulant activity < 160 IU / mg in 900 ml of distilled water at 25 DEG C., and 2.07 g of NaNO2 (97%) are added to the solution and then 5N HCl is added to pH 2.5. The reaction is monitored to detect the disappearance of nitrite using amido-iodo paper. The pH of the solution is brought to 10.5 with a concentrated solution of NaOH and NaBH4 is added starting the heparin. an amount of 1,5% by weight of

The solution is allowed to stand at ambient temperature for 20 hours and then acidified with 5N HCl, bringing the pH to 3.2 with a view to destroying excess NaBH4. Then the solution is neutralized with 5N NaOH, bringing the pH to 6.8. The solution obtained is purified by ultrafiltration, in view of the elimination of salts and substances of very low molecular weight, according to the specifications indicated by the European Pharmacopoeia, Fourth Edition, pages 999 and 1610.

The solution is then lyophilized and low molecular weight heparin is obtained in an amount of not less than 70% by weight of the starting heparin. The product obtained, without further purification, has an N-nitrosamine content below the limits of the European Pharmacopoeia, Fourth Edition (0.25 ppm), as mentioned in the description of the invention and in TABLE 1.

While the product obtained according to the depolymerization procedure of the present invention is in the European Pharmacopoeia, Fourth Edition, specification for nitrite and N-nitrosamine content, all is subjected to a purification procedure, in order to verify the efficiency of this procedure, as shown in the following examples. The purification procedure is a general procedure that can be applied in the purification of an LMWH obtained by depolymerization by nitrosation. EXAMPLE 2: Heating Purification

50 grams of LMWH, obtained as described in the example, are dissolved in 1000 ml of distilled water without 12 nitrites, and the pH is adjusted to 4.5 with 1N HCl. The solution is then heated to 50 ° C for 30 minutes with stirring. The solution is cooled and the pH is brought to 12.0 with 5N NaOH, warming to 80øC with stirring for 30 minutes. The pH is corrected with 2N HCl to a pH of 6.7. The sodium chloride is then removed from the solution by ultrafiltration using a 600 dalton cut-off membrane and bidistilled water without nitrites or nitrates. The purified heparin is lyophilized and 48.35 g of low molecular weight heparin is obtained with a final amount of 96.7%. EXAMPLE 3: Microwave Radiation Purification

70 grams of LMWH, obtained according to example 1, is added to 1400 ml of bidistilled water without nitrites or nitrates, the pH being brought to 12 with 1N NaOH. 5 g of powdered montmorillonite is added to the solution, which is then subjected to microwave irradiation at 2450 MHz for 100 seconds at 600 watts and allowed to cool to 25øC, correcting the pH with 5N HCl to 6 , 5. The inert material is filtered, purified and the solution is lyophilized; 68.7% of the product is obtained, which represents a total amount of 98.14%. EXAMPLE 4: Microwave Radiation Purification

65 grams of LMWH, obtained according to example 1, is dissolved in 800 ml of doubly distilled water without nitrites or nitrates. 4 g of powdered montmorillonite is added to the solution, brought to pH 11.0 with 2N NaOH and subjected to microwave radiation at 2450 MHz at 600 watts for 100 seconds. The solution is then cooled and the pH is brought to 6.5-7.0 with 1N HCl, the inert material is filtered and the resultant solution is subjected to ultrafiltration with the use of a cutting membrane of 600 daltons. The ultrafiltered solution is freeze-dried and yields 63.3 grams of product, i.e., 97.38%. EXAMPLE 5: Microwave Radiation Purification

The procedure of Example 4 is repeated using 60 grams of LMWH, obtained according to example 1, dissolved in 1000 ml of water. 9 grams of powdered montmorillonite is added to the solution, which is brought to pH 12 with 2N NaOH, subjecting the radiation for 60 seconds with 2450 MHz microwave at 600 watts. The pH is corrected with 2N HCl to a pH of 7.1. The inert material is filtered and the resulting solution is lyophilized after ultrafiltration to give 59.1 grams of product, i.e., 98.5%. EXAMPLE 6: Microwave Radiation Purification

The procedure of claim 4 is repeated using 65 grams of LMWH, obtained according to example 1, dissolved in 1000 ml of water. 8 grams of powdered montmorillonite is added to the solution, which is brought to pH 10.5 with 2N NaOH, subjecting the radiation for 120 seconds with 2450 MHz microwave at 600 watts. The solution is cooled to 24øC and the pH is corrected with 2N HCl, bringing it to 6.9. The inert material is filtered and the resulting solution is ultrafiltrated using a 600 dalton cut-off membrane and desalted prior to lyophilization. 64.1 grams of purified product is obtained, i.e., 98.6%. EXAMPLE 7: Preparation of low molecular weight heparin by depolymerization using sodium nitrite

20 g of sodium heparin having anticoagulant activity < 160 ul / mg in 133 ml of distilled water at 25 ° C, 0.427 g of NaNCH (97%) are added, as well as hydrochloric acid with a concentration of 5N to reach pH 2.5. During the reaction, starch-iodine paper is used to control the disappearance of the nitrite. The pH of the solution is brought to 10.5 with a concentrated solution of sodium hydroxide and 0.3 g of 1.5% NaBH 4 are added.

The solution is allowed to stand at room temperature for 20 hours and then acidified to bring the pH to 2.8 in order to destroy excess NaBH 4. The solution obtained is purified by ultrafiltration. EXAMPLE 8: Preparation of low molecular weight heparin by depolymerization using sodium nitrite

20 g of sodium heparin having anticoagulant activity < 160 ul / ml 133 ml of distilled water at 15 ° C and 0.412 g of NaNO 2 (97%), as well as 5N hydrochloric acid are added to reach a pH of 2.5. During the reaction, starch-iodine paper is used to control the disappearance of the nitrite. The pH of the solution is brought to 10.5 with a concentrated solution of sodium hydroxide and 0.3 g of 1.5% NaBH 4 are added.

The solution is allowed to stand at room temperature for 20 hours and then acidified to bring the pH to 3.0 in order to destroy excess NaBH 4. The solution obtained is purified by ultrafiltration. 15

DOCUMENTS REFERRED TO IN THE DESCRIPTION The list of documents referred to by the author of the present application was made solely for the reader's information and is not an integral part of the European patent document. Notwithstanding careful preparation, the IEP assumes no responsibility for any errors or omissions.

Patent documents referred to in the description EP 0014184 A [0004] EP 0040144 A [0004] EP 0121067 A [0004] US 4804652 A [0005] US 4351938 A [0005] US 5019649 A, JC Lormeau US 5599801 A, JF Branellec [0014]

Non-patent literature referred to in the description • L.O. ANDERSSON et al. Throm. Res., 1976, vol. 9, 575 [0003] F.A. OFOSU et al. Mechanism of action of low molecular weight heparins and heparinoids. Ballicre's Clin.

Haematol., 1990, vol. 3, 505-529 [0005] • L.BARA et al. Increased anti Xa bioavailability for low molecular weight heparin compared with unfractionated heparin. Semin. Thromb. Hemost., 1985, vol. 11, 316-317 [0005]

Lisbon, 02/02/2009

Claims (5)

A process for the preparation of a depolymerized purified heparin solution, comprising the following steps: i) preparing a low molecular weight heparin by depolymerizing a heparin with aqueous sodium nitrite and ii) treatment of the depolymerized heparin in the aqueous solution at a temperature of from 40 ° C to 90 ° C for a period of from 0.5 to 3 hours with a pH between 3.0 and 13.0 and then iii) leading to the solution at a pH of 11 to 13 and at a temperature comprised between 70 ° C and 90 ° C The process according to claim 1, wherein the solution in step ii) is brought to a pH of 12. The process according to claim 1 or 2, wherein membrane ultrafiltration is used to purify the LMWH of the solution resulting from step iii). A process for the preparation of a depolymerized purified heparin comprising the following steps: preparation of a low molecular weight heparin by depolymerization of a heparin with sodium nitrite in an aqueous solution and treatment of the depolymerized heparin in the aqueous solution with microwaves between 900 and 2450 MHz and at power between 600 and 1000 watts, for a period of time comprised between 60 and 300 seconds. The method of claim 4 wherein the depolymerized heparin is added an inert silicate, preferably a montemorilonite class silicate of the silicates. The process according to claim 5, wherein the inert silicate is added in an amount of 5 to 20% by weight, based on the weight of the heparin. The process according to claims 4 to 6, wherein the depolymerized heparin is in an aqueous solution at a concentration comprised between 1 and 25% and having a pH between 6 and 13. Lisbon, 02/02/2009